1. Field of the Invention
The disclosed invention relates to the isolation and characterization of the csa operon, which encodes the CS4 pili and its use as an immunogenic agent with utility in preventing ETEC colonization of a subject.
2. Description of the Related Art
Human ETEC strains are a major cause of diarrhea in infants and young children in developing countries (Black et al., Lancet, I: 141-143 (1981); Levine, J. Infect. Dis., 155:377-389 (1987); Qadri, et al., J. Clin. Microbiol., 38:27-31 (2000)), which account for a high rate of infantile morbidity and mortality. Human ETEC strains are also a major cause of travelers' diarrhea. (Black, Rev. Infect. Dis., 8S:S131-S135 (1986); DuPont et al., N. Engl. J. Med., 285:1520-1521 (1976); Hyams et al., N. Engl. J. Med., 325:1423-1428 (1991); Merson, et al., N. Engl. J. Med., 294:1299-1305 (1976)). ETEC infection is characterized by watery diarrhea often accompanied by low-grade fever, abdominal cramps, malaise and vomiting.
ETEC strains colonize the small bowel lumen by means of surface pili called colonization factor antigens (CFA), and coli surface antigens (CS), and cause diarrhea through the action of heat labile (LT) and/or heat stable (ST) enterotoxins. ETEC fimbriae are proteinaceous filaments exhibiting different morphologies such as rigid rod like shapes of 2-7 nm in diameter, fibrilar thin flexible wiry structures, or bundles. (Gaastra et al., Trends. Microbiol., 4:444-452 (1996)).
Human ETEC strains display a variety of over 20 serologically distinct pili on their cell surfaces. The most common human ETEC strains express CFA/I, CFA/II and CFA/IV (Levine, et al., “Fimbrial vaccines,” In P. Klemm (ed.), Fimbriae: adhesion, biogenics, genetics and vaccines, Boca Raton: CRC Press, 1994; McConnell, et al., Epidemiol. Infect., 106: 477-484 (1991)). CFA/I produces a single type of fimbriae, while CFA/II and CFA/IV strains produce several types of coli surface antigens. CFA/II strains express CS1, CS2 and CS3; and CFA/IV strains (originally called PCF8775) express the nonpilus antigen CS6 either alone or together with CS4 or CS5 fimbria. (McConnell, et al., Infect. Immun., 56:1974-1980 (1988); McConnell, et al., FEMS Microbiol. Lett., 52:105-108 (1989); Svennerholm, et al., Infect. Immun., 56:523-528 (1988); Thomas, et al., J. Gen. Microbiol., 131:2319-2326 (1985)). The occurrence of CS4+CS6+producing strains is restricted to serotype O25:H42. (McConnell, et al., Infect. Immun., 56:1974-1980 (1988); Willshaw, et al., FEMS Microbiol. Lett., 49:473-478 (1988); Willshaw, et al., FEMS Microbiol. Lett., 56:255-260 (1990); Willshaw, et al., FEMS Microbiol. Lett., 66:125-129 (1991)).
The CS4 pili is rigid, 7 nm in diameter, and is composed of subunits with a molecular mass of 17.0 kDa. (Knutton, et al., Infect. Immun., 57:3364-3371 (1989); McConnell, et al., Infect. Immun., 56:1974-1980 (1988); Wolf, et al., Infect. Immun., 57:164-173 (1989)). Because of their epidemiological importance and due to the fact that cross protection does not occur between strains of ETEC expressing different fimbriae, at least these CFA/I and CS1—CS6 fimbrial types must be included in a broad spectrum ETEC vaccine, (Gaastra et al., Trends. Microbiol., 4:444-452 (1996); Levine, J. Pediatr. Gastroenterol. Nutr., In Press (2000); Levine, et al., “Fimbrial vaccines,” In P. Klemm (ed.), Fimbriae: adhesion, biogenics, genetics and vaccines, Boca Raton: CRC Press, 1994). Of these seven important fimbriae, only the genes encoding CS4 have not been cloned and sequenced.
The genes that are required for the expression of functional pili are characteristically linked in gene clusters (Sakellaris and Scott, Mol. Microbiol., 30:681-687 (1998)), and consist of the structural genes, assembly cassette genes and regulatory genes. The assembly cassette genes include chaperone and usher genes. The chaperone protein is thought to bind to fimbrial subunit proteins in the periplasmic space and prevent premature folding and degradation. The usher proteins are outer membrane proteins that serve as pores for the transport and assembly of the fimbriae. The structural gene encodes for the pilin protein that forms the fimbriae that is composed of repeated subunits of the pilin protein. Some fimbriae such as, CFA/I, CS1 and CS2, contain a minor pilin protein which is associated with the pili tip, that is probably involved in the attachment of the bacteria to the cell receptors. (Sakellaris, et al., Proc. Natl. Acad. Sci. U.S.A., 96:12828-12832 (1999)). Fimbria expression is controlled by genes such as rns and cfaD that are similar to the araC family of transcriptional regulators that positively regulate transcription. (Grewal et al., Vaccine, 11:221-226 (1993); de Haan et al., FEMS Microbiol. Lett., 67:341-346 (1991); 19, 21, Savelkoul, et al., Microb. Pathog., 8:91-99 (1990)).
Genes encoding ETEC fimbriae have been located on large plasmids [CFA/I, (Hamers et al., Microb. Pathog., 6:297-309 (1989); 20); CS1, (Froehlich et al., Mol. Microbiol., 12:387-401 (1994)); CS3, (Jalajakumari, et al., Mol. Microbiol., 3:1685-1695 (1989), Manning, et al., Mol. Gen. Genet., 200:322-327 (1985)); CS5, (Duthy, et al., J. Bacteriol., 181:5847-5851 (1999)) and CS6, (Wolf, et al., FEMS Microbiol. Lett., 148:35-42 (1997))], or on the chromosome [CS2, (Froehlich et al., Mol. Microbiol., 12:387-401 (1994), Froehlich et al., Infect. Immun., 63:4849-4856 (1995))]. Early experiments to locate the CS4 encoding genes revealed disparate results without conclusive localization, (Sommerfelt, et al., Microb. Pathog., 11:297-304 (1991); Sommerfelt, et al., Infect. Immun., 60:3799-3806 (1992); Sommerfelt, et al., J. Clin. Microbiol., 30:1823-1828 (1992); Willshaw, et al., FEMS Microbiol. Lett., 56:255-260 (1990); Wolf, et al., Infect. Immun., 57:164-173 (1989)).
Because the attachment of ETEC strains to intestinal cells is crucial for establishment of infection, the prevention of disease is based mainly upon immune responses against the pili that interfere with the attachment process. Studies performed with 14 healthy human volunteers who ingested 5×108 E. coli E24377A (O139:H28), a CS1 and CS3 producing strain, showed that all 14 became colonized, 9 developed a typical diarrheal syndrome, and 6 of these ill persons manifested a significant increase in serum IgG antibody to purified CS1 and CS3 antigens. Levine, M. M., et al., Infect. Immun., 44:409-420 (1984). These results suggest that the fimbria play a role in pathogenesis, and stimulate an immune response.
Passive protection against ETEC infection was demonstrated in a clinical trial done at the University of Maryland, which demonstrated that oral prophylaxis with hyperimmune anti CFA/I immunoglobulin provided 90% protection against diarrhea caused by oral challenge with 109 cfu of ETEC strain H10407, a CFA/I producing strain. Tacket, et al., N. Eng. J. Med., 318: 1240 (1988). In another study oral immunization with purified CS1 and CS3 antigens encapsulated in biodegradable polymer microspheres were used to induce the development of IgA anti CS ASC (Antibody Secreting Cells) and jejunal fluid secretory IgA anti CS in 50% of the vaccinees. Levine, et al., Fimbriae (pili) adhesions as vaccines, in Protein-Carbohydrate Interactions in Biological Systems. The molecular Biology of Microbial Pathogenicity, Lark, et al., Eds., Academic Press, London, p 154, 1986. This exposure also protected 30% of vaccinees from diarrhea following challenge with the virulent ETEC E24377A (CS1+CS3+LT+ST+) strain. A protective efficacy of 75% was demonstrated by immunization with the attenuated ETEC strain. Feeding volunteers with 5×1010 live E. coli E1392-75-2A (O6:H16) a CS1+ CS3+ LT− ST− strain, induced significant rise in intestinal fluid secretory IgA antibodies to CS1 and CS3 fimbria, and conferred protection to 9/12 volunteers that were challenged with a virulent heterologous serotype strain ETEC E24377A (O139:H28).
Another approach to develop an ETEC vaccine is to immunize with killed mixed ETEC strains. This type of vaccine is based on the fact that prior infection with an ETEC strain elicits protective immunity against a clinical illness that might be caused from subsequent exposure to the homologous strain. Oral immunization of children and adult volunteers with such a vaccine resulted in significant intestinal IgA responses against the CFA and CS components of the various strains. The vaccine induced high level of intestinal IgA antibody, IgA antibody ASC in the blood, and serum antibodies towards the colonization factor antigens. Jertborn, et al., Vaccine 16:255 (1997). Those results indicate that antibodies towards ETEC pilis provide protection against diarrhea caused by ETEC strains.
A combined vaccine against diarrheal disease caused by Shigella, Salmonella and ETEC has been proposed. These vaccines are composed of live attenuated Shigella or Salmonella strains that express ETEC fimbriae. The recombinant bacteria colonize the intestine and induced the mucosal and systemic immune response against the bacteria and the pili. Noriega et al., (Infect. Immun., 64:23-27 (1996)) co-expressed CFA/I and CS3 in Shigella CVD1203, immunized guinea pigs and mice, and showed that the immunized animals developed high titer of tears secreted IgA (similar to mucosal sIgA), and serum IgG antibodies, towards the Shigella LPS and the fimbrial antigens. The ETEC human recombinant LT (K63) gene was expressed in Shigella CVD1204, a guanine dependent strain, and showed production of sIgA and serum IgG toward the Shigella LPS as well as towards the LT-A and LT-B subunits of the LT enterotoxin, following immunization of guinea pigs. Koprowski, et al., (Infect. Immun., 68:4884-92 (2000)) co-expressed in CVD1204 the CFA/I and LT (K63) antigens, and demonstrates production of antibodies of the sIgA and serum IgG toward the Shigella LPS, the CFA/I pili antigens and the LT antigens. Altboum et al, (Attenuated Shigella flexneri 2a ΔguaBA strain CVD 1204 expressing ETEC CS2 and CS3 fimbriae as a live mucosal vaccine against Shigella and enterotoxigenic Escherichia coli infection, In press) immunized guinea pigs with a mixture of CVD 1204 strains expressing ETEC CS2 and CS3 fimbria. All the immunized animals developed tears sIgA and serum IgG antibodies against Shigella LPS, CS2 and CS3 antigens, agglutinating antibodies against Shigella and ETEC CS2 and CS3 strains, and were protected against keratoconjunctivitis caused by eye challenge with the virulent S. flexneri 2a 2457T strain. Those results indicate that a combined immunization with live attenuated Shigella strains expressing ETEC fimbria might induce protection against Shigellosis and ETEC infection.